Dopamine transporter knockout mice

ABSTRACT

A recombinant rodent comprises cells containing a pair of genomic dopamine transporter protein alleles, wherein at least one of said alleles is incapable of expressing endogenous dopamine transporter protein. The rodent may be a homozygote, where both of said alleles are incapable of expressing endogenous dopamine transporter protein, or the rodent may be a heterozygote, and one of said alleles expresses endogenous dopamine transporter protein. The rodent is preferably a mouse.

This invention was made with Government support under grant numberNS19576 from the National Institutes of Health. The Government hascertain rights to this invention.

This application claims the benefit of U.S. provisional application No.60/004,695, filed on 02 Oct., 1995.

FIELD OF THE INVENTION

This invention relates to methods of increasing spontaneous motoractivity in a subject in need of such treatment, particularly subjectsafflicted with Parkinson's disease or tardive dyskinesia.

BACKGROUND OF THE INVENTION

Dopamine is involved in the control of motor function, cognition andaffect (A. Dahlstrom and K. Fuxe, Acta Physiol. Scand. 232, 1-55 (1965);U. Ungerstedt, Acta Physiol. Scand. 367, 1-48 (1970)). Imbalance in thedopamine system is believed to be involved in such conditions asschizophrenia (T. Crow, Brit. J. Psychiatry 137, 383-386 (1980);Fibiger, in : The Mesolimbic dopamine system : From motivation toaction, 615-37 (Eds. P. Willner and J. Scheel-Krger 1991)), Parkinson'sdisease (H. Ehringer and O. Hornykiewitz, Klin. Wochenschr. 38,1236-1239 (1960), tardive dyskinesia and drug addiction (R. Wise and P.P. Rompre, Ann. Rev. Psychol. 40, 191-225 (1989); G. Koob and F. Bloom,Science 242, 715-723 (1988); G. DiChiara et al., Proc. Natl Acad. Sci.U.S.A. 85, 5272-5278 (1998)).

The dopamine transporter, a member of the large family of Na⁺ /Cl⁻dependent transporters, aids in terminating dopaminergicneurotransmission by rapid reuptake of dopamine (B. Giros and M. Caron,Trends Pharmacol. Sci. 14, 43-49 (1993). It is a major target of thepsychostimulant drugs cocaine and amphetamine (B. Giros and M. Caron,Trends Pharmacol. Sci. 14, 43-49 (1993); M. Ritz et al., Science 237,1219-1223 (1987)), but its overall role in vivo is still poorlyunderstood.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a recombinant rodentcomprising cells containing a pair of genomic dopamine transporterprotein alleles, and wherein at least one of said alleles is incapableof expressing endogenous dopamine transporter protein. The rodent may bea homozygote., where both of said alleles are incapable of expressingendogenous dopamine transporter protein, or the rodent may be aheterozygote, and one of said alleles expresses endogenous dopaminetransporter protein.

A further aspect of the present invention is a method of upregulatingspontaneous motor activity in a subject in need of such treatment. Themethod comprises inhibiting dopamine transporter function (i.e.,dopamine reuptake mediated by the dopamine transporter protein) in thesubject by an amount effective to enhance spontaneous motor activity inthat subject. The method may be carried out by any suitable means, suchas by administering a dopamine transporter blocker to said subject in anamount effective to enhance spontaneous motor activity. Suitablesubjects include those afflicted with Parkinson's disease and thoseafflicted with tardive dyskinesia, particularly drug-induced tardivedyskinesia.

A still further aspect of the present invention is the use of a dopaminetransporter inhibitor for the preparation of a medicament for enhancingspontaneous motor activity in that subject, as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the targeting of the mouse DAT gene in ES cells and mice.Gene structures and restriction maps for the DAT targeting construct(pD7H15/Neo), wild-type and recombinant are shown. Black and open boxesrepresent coding and untranslated exons, respectively. Arrow linesindicate the EcoRI restriction fragment sizes for wild-type andrecombinant DNA. Restriction sites are : C, ClaI; E, EcoRI; H, HindIII;X, XbaI. The probe used for Southern analysis is shown as a thick blackline.

DETAILED DESCRIPTION OF THE INVENTION

We herein describe the first gene inactivation of a member of the Na⁺/Cl⁻ -dependent transporter family. We provide direct in vivo evidencethat the dopamine transporter may represent the most crucial functionaldeterminant of neurotransmitter tone in the central nervous systemidentified to date. The functional ablation of the dopamine transporterin the mouse produces a phenotype in which homozygote animals display amarked increase in spontaneous locomotor activity. This phenotype ispresumably a reflection of the prolonged availability of dopamine at thesynapse, as effects of similar magnitude are observed following completeblockade of the transporter with maximal doses of psychostimulants (G.DiChiara and A. Imperato, Proc. Natl Acad. Sci. U.S.A. 85, 5272-5278(1998); B. Giros and M. Caron, Trends Pharmacol. Sci.14, 43-49 (1993);M. Ritz et al., Science 237, 1219-1223 (1987); M. Jaber et al.,Neuroscience, 65, 1041-1050 (1995)). This hyperlocomotion phenotype isapparent even in light of major adaptive decreases in the mRNAs for theD1 and D2 receptors, the main targets of synaptic dopamineresponsiveness. This phenotype is even more impressive if one considersthat levels of dopamine may be markedly lower in homozygote animals.Indeed, as seen by immunoblotting and immunohistochemistry, tyrosinehydroxylase, the rate limiting enzyme of dopamine synthesis, is reducedbymore than 80%. in dopamine neurons and projections. In addition, theobservation that heterozygote animals have certain characteristics whichare intermediate between wild type and homozygote mice, reinforces thenotion of a fundamental role for the dopamine transporter in themaintenance of normal dopaminergic neurotransmission.

The properties of the DAT knockout mice make them an attractive animalmodel for the study and development of drugs for the management ofdopaminergic dysfunction. However, the most exciting consequences ofthesedata are in the development of new therapeutic strategies forcommon neurological disorders. For example, these data indicate thattargeting ofthe DAT with high affinity antagonists (J. Boja et al. Mol.Pharmacol. 47, 779-786 (1995)) in illnesses such as Parkinson's disease,where the effective levels of dopamine are markedly decreased, (H.Ehringer and O., Hornykiewitz, Klin. Wochenschr. 38, 1236-1239 (1960))have beneficial clinical consequences.

Our results further demonstrate that the DAT, in addition to itsproposed role in the reinforcing properties of the psychostimulantscocaine and amphetamine, (M. Ritz et al. Science 237, 1219-1223 (1987))represents theprimary target for their locomotor effects. Since cocaine,and to a lesser extent amphetamine, interact with other monoaminetransporters, the DAT homozygote and heterozygote animals should provean excellent model to elucidate complex behavioral paradigms such asreward, addiction and tolerance properties of these drugs.

Animals suitable for carrying out the present invention are, in general,rodent species. Rat and mouse are preferred, and the mouse is mostpreferred. Animals in every stage of development, including juvenile,adolescent, and adult, are included in this description, with adultanimals particularly preferred.

"Knockout" animals refers to animals whose native or endogenous DATallele or alleles have been disrupted by homologous recombination andwhich produce no functional DAT of their own. Knockout animals may beproduced in accordance with techniques known in the art, particularly bymeans of in vivo homologous recombination, M. Capechi, Science 244,1288-1292 (1989), in light of the known sequence for DNA encoding theDAT. See, e.g., A. Eshleman et al., Molec. Pharmacol. 45, 312-316(1994). DAT knockout animals of species other than mice can be producedby variation of the procedures carried to produce Apo E knockout micethat will be apparent to those skilled in the art.

Animals described in the present invention are animals having at leastone allele incapable of expressing endogenous dopamine transporterprotein (DAT), and include both the heterozygous or homozygous forms(e.g., DAT+/-and DAT -/-). Animals may be chimeric animals or animals inwhich essentially all cells are recombinant. Preferably at least thebrain cellsare recombinant.

Animals of the present invention are useful in a variety of screeningassays, as discussed below. In such assays, the compound being screenedmay be administered to the animal by any suitable means, includingorally and parenterally (e.g., by subcutaneous injection). Theparticular activity detected in the animal (i.e., neuroleptic,antischizophrenic, control of substance abuse, neuropharmacological,appetite modulating, aggressive behavior modulating, and addictiveactivities) may be detected in accordance with techniques known to thoseskilled in the art of neuropharmacology.

A method of screening a compound for neuroleptic activity comprisesadministering a test compound to a recombinant animal as given above,and then detecting the presence or absence of neuroleptic activity insaid animal. The method is particularly useful wherein the neurolepticactivityis antischizophrenic activity.

A method of screening a compound for activity for the control ofsubstance abuse (i.e., inhibiting or treating addictive behavior)comprises administering a test compound to a recombinant animal(preferably homozygous) as given above, and then detecting the presenceor absence of activity for the control of substance abuse in the animal.Substance abuses such as amphetamine abuse and cocaine abuse (i.e.,amphetamine craving and cocaine craving) are particularly preferredcategories of substance abuse for which compounds useful for thetreatment thereof may be screened by the instant method.

A method useful as a negative control for screening a compound fornorepinephrine or serotonin transporter activity comprises administeringatest compound to a recombinant animal (preferably homozygous) as givenabove, and then detecting the presence or absence ofneuropharmacological activity for the compound in the animal.

A method of screening a compound for appetite modulating activitycomprisesadministering a test compound to a recombinant animal(preferably homozygous) as given above, and then detecting the presenceor absence of appetite modulating activity in the animal. The activityscreened for may be either activity in increasing or decreasingappetite.

A method of screening a compound for aggressive behavior modulatingactivity comprises administering a test compound to a recombinant animal(preferably homozygous) as given above, and then detecting the presenceorabsence of aggresive behavior modulating activity in the animal. Theactivity screened for may be either activity in increasing or decreasingaggressive behavior.

A method of screening a compound for substance abuse potential (i.e.,reinforcing activity) comprises administering a test compound to aheterozygous recombinant animal as given above, and then detecting thepresence or absence of addictive behavior with respect to the compoundin the animal (i.e., the finding that the animal develops a craving forthat compound, or that the administration of that compound is a positivereinforcement for that animal). The presence of addictive behaviorindicating the compound has a potential for substance abuse.

As noted above, a further aspect of the present invention is a method ofupregulating spontaneous motor activity in a subject in need of suchtreatment. The method may be carried out on human subjects or on animalsubjects for veterinary purposes. Suitable subjects include thoseafflicted with Parkinson's disease and those afflicted with tardivedyskinesia, particularly drug-induced tardive dyskinesia. A variety ofdopamine transporter inhibitors (also called dopamine uptake inhibitors;herein referred to as active compounds) of diverse structure are known.See, e.g., S. Berger, U.S. Pat. No. 5,217,987; J. Boja et al., Molec.Pharmacol. 47, 779-786 (1995); C. Xu et al., Biochem. Pharmacol. 49,339-50 (1995); B. Madras et al., Eur. J. Pharmacol. 267, 167-73 (1994);F.Carroll et al., J. Med. Chem. 37, 2865-73 (1994); A. Eshleman et al.,Molec. Pharmacol. 45, 312-16 (1994); R. Heikkila and L. Manzino, Eur. J.Pharmacol. 103, 241-8 (1984). Dopamine transporter inhibitors are, ingeneral, ligands that bind in a stereospecific manner to the dopaminetransporter protein. Examples of such compounds are:

(1) tricyclic antidepressants such as buprion, nomifensine, andamineptin;

(2) 1,4-disubstituted piperazines, or piperazine analogs, such as 1- 2-bis(4-fluorophenyl) methoxy!ethyl!-4-(3-phenylpropyl)piperazinedihycrochloride (or GBR 12909), 1- 2- bis(phenyl)methoxy!ethyl!-4-(3-phenylpropyl)piperazine dihydrochloride (orGBR12935),and GBR13069;

(3) tropane analogs, or (disubstituted phenyl) tropane-2 beta-carboxylicacid methyl esters, such as 3β-(4-fluorophenyl)tropane-2β-carboxylicacid methyl ester (or WIN 35,428) and3β-(4-iodophenyl)tropane-2β-carboxylic acid isopropyl ester (RTI-121);

(4) substituted piperidines, or piperidine analogs, such as N-1-(2-benzo b!-thiophenyl) cyclohexyl! piperidine, indatraline, and 4- 2-bis(4-fluorophenyl)methoxy!ethyl!-1-(3-phenylpropyl) piperidine (orO-526);

(5) quinoxaline derivatives, or quinoxaline analogs, such as7-trifluoromethyl-4-(4-methyl-1-piperazinyl)pyrrolo 1,2-α!quinoxaline(or CGS 12066b); and

(6) other compounds that are inhibitors of dopamine reuptake, such asmazindol, benztropine, bupropion, phencyclidine, methylphenidate, etc.

Because the DNA sequence of the dopamine transporter is known (See,e.g., A. Eshleman et al., supra), this sequence can be used to inhibitor downregulate dopamine transporter in animals by means ofadministering to the subject antisense oligonucleotides that bind tomRNA encoding the dopamine transporter in an amount effective to inhibitthe function of thedopamine transporter, or by means of inhibitingdopamine transporter transcription by administering oligonucleotidesthat specifically bind to DNA encoding the dopamine transporter, and, byhomologous recombination, inhibit the activity of the dopaminetransporter.

The active compounds disclosed herein can be prepared in the form oftheir pharmaceutically acceptable salts. Pharmaceutically acceptablesalts are salts that retain the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; and salts formed with organicacids such as, forexample, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid,benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, ptoluenesulfonic acid, naphthalenedisulfonic acid,polygalacturonic acid, and the like; (b) saltsformed from elementalanions such as chlorine, bromine, and iodine, and (c)salts derived frombases, such as ammonium salts, alkali metal salts such as those ofsodium and potassium, alkaline earth metal salts such as thoseof calciumand magnesium, and salts with organic bases such as dicyclohexylamineand N-methyl-D-glucamine.

Pharmaceutical compositions for use in the present method of increasingspontaneous motor activity include those suitable for inhalation, oral,rectal, topical, (including buccal, sublingual, dermal and intraocular)parenteral (including subcutaneous, intradermal, intramuscular,intravenous and intraarticular) and transdermal administration. Thecompositions may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art. Theformulations mayconveniently be presented in unit dosage form and may beprepared by any ofthe methods well known in the art.

The dose of active compound will vary according to the the particularcompound administered, the route of administration, the manner offormulation, the condition of the subject, and the dose at which adversepharmacological effects occur. One skilled in the art will take suchfactors into account when determining dosage. Illustrative examples oftypical dosages are, for methylphenidate, from 1 or 10 to 60 or 100 mgpersubject daily, and for buproprion, from 10 or 100 to 450 or 1000 mgper subject daily.

In the manufacture of a medicament according to the invention (a"formulation"), active agents or the physiologically acceptable saltsthereof (the "active compound") are typically admixed with, among otherthings, an acceptable carrier. The carrier must be acceptable in thesenseof being compatible with any other ingredients in the formulationand must not be deleterious to the patient. The carrier may be a solidor a liquid,or both, and is preferably formulated with the compound as aunit-dose formulation, for example, a tablet, which may contain from0.5% to 99% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention(e.g., the formulation may contain one or more additionalanti-tubercular agents as noted above),which formulations may beprepared by any of the well known techniques of pharmacy consistingessentially of admixing the components, optionally including one or moreaccessory therapeutic ingredients.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as asolution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, theformulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder. Formulations for oral administration may optionally includeenteric coatings known in the art to prevent degradation of theformulation in the stomach and provide release of the drug in the smallintestine.

Formulations suitable for buccal (sublingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundinan inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender theformulation isotonic with the blood of the intended recipient.Aqueous and non-aqueous sterile suspensions may include suspendingagents and thickening agents. The formulations may be presented inunit\dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,saline or water-for-injection immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets of the kind previously described.

Formulations suitable for rectal administration are preferably presentedasunit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable fortransdermal administration may also be delivered byiontophoresis (see, e.g., Pharmaceutical Research 3, 318 (1986)) andtypically take the form of an optionally buffered aqueous solution ofthe active compound.

Those skilled in the art will appreciate numerous variations which maybe made to the foregoing in carrying out the instant invention, whichwill bereadily apparent from the literature concerning recombinantanimals. See, e.g., H. Chen et al., PCT Application WO 95/03397; H. Chenet al., PCT Application WO 95/03402; C. Wood et al., PCT Application WO91/00906; C. Lo et al., PCT Application WO 90/06992; P. Leder et al.,U.S. Pat. No. 4,736,866; and T. Wagner et al., U.S. patent applicationNo. 4,873,191.

The present invention is explained in greater detail by the followingExamples. These examples are illustrative of the present invention, andare not to be construed as limiting thereof.

EXAMPLE 1

Targeting of the Mouse Dopamine Transporter

This example describes the production, by means of in vivo homologousrecombination, of a strain of mice in which the gene encoding thedopaminetransporter (DAT) has been disrupted.

A rat DAT cDNA probe comprising the N-terminal to the thirdtransmembrane domain (B. Giros et al., FEBS Letters 295, 149-154 (1991))was used to screen a mouse 129 Sv/J genomic library in accordance withknown techniques (B. Giros et al., Nature 342, 923-926 (1989)). A 12 kbphage containing the first three exons of the mouse DAT gene wasisolated, and a7.5 kb HindIII fragment (from an internal site to a phagecloning site) wasexcises and subcloned into pGEM4Z. A EcoRI-SAlI-NotIcassette containing the neomycin resistance (ncor) gene under thecontrol of the PGK promotor was introduced into the NotI site of theconstruct to provide pD7H15/neo. The neor cassette was cloned in theopposite direction as the DAT gene, and DNA sequencing was used toinsure that pD7H15/neo did not contain a consensus start site that couldbe used to resume DAT translation. pD7H15/neo was linearized with AatIIprior to electroporation into ES cells. Cell culture, electroporation ofE14TG2a ES cells (M. Hooper et al., Nature 326, 292-295 (1987)) andgeneration of chimeric mice were performed in accordance with knowntechniques (B. Kohler and O. Smithies, Proc. Natl. Acad. Sci. U.S.A. 86,8932-8935 (1989)). For Southern analysis, 5-15 μg DNA from ES cells ortail biopsies were digested withEcoRI and HindIII and probed with a 1.1kb genomic DNA probe, and a neor probe to ensure that there was only onecopy of the construct in ES cells.About 20%. of the tested ES cells werepositive for homologous recombination, and three clones were selectedfor karyotyping and injection into C57/B6J blastocysts. Chimeric maleswere mated with C57/B6Jfemales, and agouti coat pups were typed bySouthern blotting of tail biopsies. Two germ-line transmitting males(from different ES clones) wereused as colony founders. For in situhybridization, mice were sacrificed bydecapitation and the brains weredissected out, immersed overnight in 1% paraformaldehyde andcryoprotected in 15% sucrose-PBS buffer for 6-8 hours. The brains werethen frozen in liquid nitrogen and cut into frontalsections (12μm) whichwere stored at -80° C. until required. The in situ hybridizationprocedure was performed with oligonucleotide probes,labeled by tailingwith ³⁵ S! dATP (NEN), with a specific activity of2×10⁹ cpm/μg aspreviously described (M. Jabar et al., Mol. Brain Res., 23, 14-20(1994); F. Tison et al., Neurosci. Lett. 166 48-50 (1994)). Sectionswere exposed at room temperature to X-ray films (Kodak X-Omat) for 7days. Autoradiograms were scanned (Howtek Scanner) at 1200 dpi andanalyzed (NIH-Image). Probe concentration and exposure times were chosenin order to stay within a linear range of detection. Experiments wereperformed in duplicate, and comparisons were made between groups usingthe Student's t test with the control value (DAT^(+/+)) set equal to 1,and DAT^(+/-) and DAT^(-/-) values expressed relatively to it.Fordopamine uptake experiments, two striata from one mouse were homogenizedin 0.5 ml of 0.3M sucrose and synaptosomes were prepared as described(C. Pifl et al., J. Neurosci. 13, 4246-4253 (1993)). The synaptosomalpreparation (20-25 μg protein/tube) was incubated with 0.25 μCi of ³ H!dopamine (51 Ci/mmol), and increasing concentrations of dopamine (0.3μM) for 5 min. at 37° C. in a final volume of 0.5 ml uptake buffer (4 mMTrisHCI, 6.25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl₂, 1.2 MmMgSO₄, 5.6 mM D-glucose, 0.5 mM ascorbic acid, pH, 7.1). Experimentswere done in triplicate for each concentration, and non-specific uptakewas determined in the presenceof 30 μm nomifensine. Uptake was stoppedand counted as described (C. Pifl et al., J. Neurosci. 13, 4246-4253(1993)). K_(M) and Vmax were calculated by the iterative curve-fittingprograms EBDA and LIGAND, (G. McPherson, J. Pharmacol. Methods 14,213-228 (1985)) and resulting values of three separate mice per groupwere averaged.

The homozygote (DAT^(-/-)) mice showed the expected pattern of genedisruption as established by Southern blotting. No mRNA encoding the DATwas detected in the dopamine neurons of the ventral tegmental area (VTA)and the substantia nigra compacta (SNC) of homozygote animals asdemonstrated by in situ hybridization. Striatal synaptosomalpreparations from DAT^(-/-) mice show significant dopamine uptake ascompared to wild type and heterozygote (DAT^(+/-)) animals.Interestingly, the heterozygote mice show a substantial decrease in theDAT mRNA (70% approx.) and a decrease in dopamine uptake (30% approx.).These data demonstrate that we have efficiently impaired the dopamineuptake activityin DAT homozygote mice. Furthermore, it appears thatdespite the absence ofexpression of the DAT in homozygote animals eversince early embryonic stages there is no compensatory expression ofother monoamines transporters, such as norepinephrine and serotonin, andthat these transporters have no or insignificant participation in normaldopamine uptake in the basal ganglia.

EXAMPLE 2

Weight Gain and Development of Treated Animals

Homozygotes are viable but gain weight more slowly than heterozygote andwild type (DAT^(+/+)) mice. They show a significant propensity forpremature death when compared to wild type and heterozygotes. These twofindings may be linked to an impairment in food intake, as blockade oftheDAT following cocaine treatment has been associated with modificationof dietary habits (R. Byck and C. VanDyke, in: NIDA monograph 13, 97-118(1977)). DAT^(-/-) mice are fertile and mate, but the females usuallyhave an impaired maternal behavior, as offspring have to be transferredtofoster mothers in order to be successfully raised to adulthood. Thisobservation is consistent with the known role of dopamine on pituitarygland function and supports the controversial notion that the DAT playsa role in the regulation of the tuberoinfundibular dopamine pathway (B.Meister amd R. Elde, Neuroendocrinology 58, 388-395 (1993); M. BaumannandR. Rothman, Brain Res. 608, 175-179 (1993)). It may also be relatedto general modifications of cognitive or affective functions underdopaminergic influence. Previous reports indicate that cocainetreatment, which blocks the DAT, significantly modifies maternalbehavior in rats (C.Kinsley et al., Pharmacol. Biochem. Behav. 47,857-864 (1994); B. Zimmerberg and M. Gray, Physiol. Behav. 52, 379-384(1992)).

EXAMPLE 3

Spontaneous Motor Activity in Treated Animals

The DAT is believed to play a major role in the control of locomotorbehavior by regulating dopaminergic tone in the basal ganglia. Indeed,drugs that interfere with dopamine re-uptake, such as thepsychostimulantscocaine and amphetamine, are know to increase locomotorbehavior (A. Dahlstrom and K. Fuxe, supra; G. DiChiara and A. Imperato,supra; B. Girosand M. Caron, supra; M. Ritz et al., supra; M. Jaber etal., Neuroscience, 65, 1041-1050 (1995); P. Kelly et al., Brain Res. 94,507-522 (1975)) accordingly, locomotor activity in DAT mice prepared asdescribed in Example 1 above was investigated.

Mice were maintained in standard housing conditions (12 hr light/darkcycle) and were allowed free access to food and water. They were housedingroups of 6 and used between the age of 6-10 weeks. Locomotor activitywas measured in Plexiglass boxes (217×268×104 mm) with 2 photocellbeamslocated across the long and the short axis respectively, 15 mm above thefloor. Activity boxes were localized in dark cabinets (6 boxes/cabinet,6cabinets) in a quiet room, and the 3 groups of mice were always analyzedat the same time. Placement of the mice and recording of the activitynumbers were done in double-blind fashion. Saline solution, cocaine (40mg/kg) or d-amphetamine (10 mg/kg) were injected by the intra-peritonealroute (10ml/kg). Statistical analysis were performed using the Student'st test.

We find that the spontaneous locomotor activity of naive homozygotes(i.e. non-habituated to the test) is highly elevated . They have ahalf-time of habituation twice as long (90 min. v. 40 min.) as the wildtype or heterozygote mice, and are 5 to 6 times more active than wildtype during both phases of the light-dark cycle.

The heterozygotes are consistently more active that the wild typeanimals but this increase is of marginal significance (P=0.06) duringthe dark phase of the cycle. On the other hand, neither theheterozygotes nor the homozygotes exhibit a significantly enhanced levelof spontaneous verticalizations or stereotypics (sniffing, grooming orrearing). It is well established that psychostimulants like amphetaminecause stereotyped behaviors by increasing extracellular dopamine in thedorsal striatum and enhance locomotor activity by increasing dopamine inthe nucleus accumbens(P. Kelly et al., supra). It is interesting to notethat DAT levels are substantially lower in the accumbens as opposed tothe striatum in rats (J. Marshall et al., J. Neuroscience 37, 11-21(1990)). Therefore, the observation of hyperlocomotion in the absence ofstereotyped behavior in homozygote animals may reflect a regionaldifference in adaption to increased dopamine tone.

Since homozygotes lack one of the major targets of psychostimulant drugsweinvestigated the locomotor effect of treatment with either cocaine oramphetamine. We found that i.p. injections of high doses of cocaine (40mg/kg) and d-amphetamine (10 mg/kg) have no significant effects onDAT^(-/-) mice. On wild type and heterozygote mice, treatment witheither of these two psychostimulants produces a 6 to 8 fold rise intheir locomotor activity; a level that is not significantly differentfrom the spontaneous activity in the homozygote mice. These findingsdirectly demonstrate the major role of the DAT in the locomotor effectsof psychostimulants, and rule out the participation of other monoaminetransporters in these effects.

EXAMPLE 4

Effect on Genes Known to Respond to an Increase in Dopamine Levels

Dopamine exerts a modulatory control on dopaminergic and dopaminoceptivecells at the pre- and post-synaptic level (M. Jaber et al.,Neuroscience, 65, 1041-1050 (1995); M. Jabar et al., Mol. Brain Res.,23, 14-20 (1994); C. Gerfen et al., J. Neurosci. 11 1016-1031 (1991); C.Gerfen et al., Science, 250, 1429-1432 (1990)). An increase indopaminergic transmission following psychostimulant treatment inducesmodifications in gene expression in cells known to be under dopaminergiccontrol (M. Jaber et al., supra). Given the dramatic increase inspontaneous locomotor activityobserved in the homozygote mice, weexamined whether this hyperactivity correlated with changes in theexpression of genes known to respond to an increase in dopamine levels.

In the basal ganglia, preproenkephalin A (PPA), which is co-expressedwith D2 receptors, (C. LeMoine et al., Science 250, 1429-1432 (1990)) isunder inhibitory influence of DA (C. Gerfen et al., J. Neurosci. 111016-1031 (1991); F. Tang et al., Proc. Natl. Acad. Sci. U.S.A. 80,3841-3844 (1983)) whereas substance P (SP) and dynorphin (Dyn), whichare coexpressed with D1 receptors (C. Gerfen et al., Science, 250,1429-1432 (1990); C. LeMoine et al., Proc. Natl. Acad. Sci. U.S.A. 88,4205-4209 (1991)), are under the excitatory influence of DA. (C. Gerfenet al. J. Neurosci. 11 1016-1031 (1991)).

By in situ hybridization (Table 1), we show that in homozygote mice PPAmRNA levels were greatly reduced (66%). mRNA coding for Dyn wasincreased by 50% while SP mRNA levels were not significantly modified.The finding that Dyn and SP mRNAs are not both up-regulated suggeststhat different activation pathways may exist for their respective genes.At the receptor level, we find that the mRNA coding for D1 and D2receptors, the two majordopamine receptors in this region, weredown-regulated by 55% and 45% respectively. This simultaneous decreasein D1 and D2 receptor mRNAs is unprecedented, as neither lesions norpharmacological and behavioral manipulations of dopamine transmissionhave ever been shown to display a down-regulation of both mRNAs. We alsodetected a 50% decrease in D2 receptor gene expression in dopamine cells(SNC and VTA), indicating a putative down-regulation of D2 autoreceptorsknown to regulate dopamine levels and firing. For each of theinvestigated mRNAs, the heterozygotes always displayed intermediatevalues between the wild type and the homozygote mice (Table 1).

                  TABLE 1    ______________________________________    mRNA quantification of in situ hybridization    autoradiograms obtained for various genes under dopamine influence    in the Striatum and ventral midbrain (VM) of Wild Type (WT),    DAT(+/-) and DAT (-/-) Mice.    Region  Gene   WT        DAT(+/-)  DAT(-/-)    ______________________________________    Striatum            PPA    1 ± 0.08                             0.83 ± 0.07*                                       0.33 ± 0.05***            SP     1 ± 0.08                             0.96 ± 0.07                                       1.17 ± 0.1            dyn    1 ± 0.07                             1.37 ± 0.09**                                       1.48 ± 0.09***            D.sub.1 R                   1 ± 0.08                             0.66 ± 0.06**                                       0.44 ± 0.04***            D.sub.2 R                   1 ± 0.09                             0.74 ± 0.07**                                       0.56 ± 0.05***    VM      DAT    1 ± 0.08                              0.31 ± 0.05***                                       ND            D.sub.2 R                   1 ± 0.02                             0.74 ± 0.05*                                       0.52 ± 0.06***    ______________________________________    Six mice per group were analyzed (methods in FIG. 1), and experiments     performed in duplicate or triplicate. Values were compared using the    Student's t test:    *: P < 0.05;    **: P < 0.01;    ***: P < 0.0001;    ND, Not Detectable.

The changes in gene expression that we observed are reflective of afunctional increase in dopaminergic neurotransmission and correlate withthe marked increase in spontaneous locomotor activity. Moreover, theseobservations show that the dopamine system is able to undergo profoundgradual adaptation and plasticity to an extent that has not beenpreviously observed with pharmacological manipulations.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

That which is claimed is:
 1. A recombinant mouse whose cells contain apair of genomic dopamine transporter protein alleles,wherein bothalleles are disrupted and do not express endogenous dopamine transporterprotein; and wherein said mouse has a phenotype of increased spontaneousmotor activity as compared to wild-type mice.
 2. A method of screening acompound for appetite modulating activity, comprising:administering atest compound to a recombinant mouse according to claim 1; and thendetecting the presence or absence of appetite modulating activity insaid mouse.
 3. A method according to claim 2, wherein said activity isincreased appetite.
 4. A method according to claim 2, wherein saidactivity is decreased appetite.
 5. A method of screening a compound foraggressive behavior modulating activity, comprising:administering a testcompound to a recombinant mouse according to claim 1; and then detectingthe presence or absence of aggresive behavior modulating activity insaid mouse.
 6. A method according to claim 5, wherein said activity isincreased aggresive behavior.
 7. A method according to claim 5, whereinsaid activity is decreased aggressive behavior.